Detection of the activation of cellular kinases associated with oncogenic signaling can serve as a valuable molecular marker for cancer diagnosis and as a predictive tool for selection of therapy. In human squamous cell carcinoma of the head and neck (SCCHN), EGFR activation is associated with therapeutic resistance, increased metastasis and poor outcomes. Histology and tumor architecture provide complementary and critical information about cancer stage and grade. By adapting and extending emerging technologies for kinase sensor biochips, we propose to develop the capability to image the distribution of cancer signaling in tumor tissue obtained by biopsy or surgical excision. We and others have developed robust, sensitive and specific biochip-based assays for kinase activity in cellular lysates, using immunodetection, radionuclide incorporation or MALDI-TOF MS analysis as a read-out. We now intend to adapt these methods to create multiplexed assays whereby multiple peptides, serving as specific substrates for kinase involved in oncogenic signaling, will be linked reversibly to the surface of a biochip. This multiplexed biosensor will be exposed to thick sections of tumor biopsies by "tissue print" to allow phosphorylation of the peptides. The cellular material will be washed away and the biochip will be interrogated by MALDI-TOF MS imaging to detect relative peptide phosphorylation, creating an image of the multiple kinase activities across the tumor section. These kinase activity images can be correlated with conventional histology and immunocytochemistry to enhance diagnosis and prognosis. To establish proof-of-principle in the R21 phase, methods will be developed using human SCCHN tissue culture cells and xenograft tumors of grown in athymic nude mice. We will take advantage of the activated EGFR kinase characteristic of this tumor and the availability of the specific EGFR inhibitors gefitinib and erlotinib to develop and validate our biochip sensor and imaging capabilities. We anticipate achieving sufficient sensitivity and resolution to detect SCCHN tumor islands embedded in extensive stroma.